Method of preparation and use of coconut water in mamalian tissue nourishment growth and healthy maintenance

ABSTRACT

A nutritional, cosmetic and pharmaceutical compositions containing active ingredients from the liquid endosperm of fresh green coconuts, and a method for obtaining the active ingredients.

This application claims priority to provisional application No.60/248,223 filed on Nov. 15, 2000.

FIELD OF THE INVENTION

The invention relates to nutritional, cosmetic and pharmaceuticalcompositions containing active ingredients from fresh green coconuts,and methods for obtaining the compositions.

BACKGROUND OF THE INVENTION

Coconut water is the liquid endosperm of Cocos nucifera L an is used asa supplement in media for the growth of plant tissue cultures. Thecoconut fruit is unique in that it accumulates large amounts of thisliquid over periods of a year or more in its life cycle. The greatestamount of coconut water is found in young, green coconuts and providesnourishment for the growth of the solid endosperm (coconut meat) insidethe hard shell of the fruit. When the fruit matures, both the solidendosperm and the remaining coconut water serve as nutrients for thedeveloping embryo and seedling. Thus coconut water serves as a naturalreservoir of nutrients to promote tissue growth¹.

The nutritional composition of coconut water obtained from fruits atdifferent stages of maturity has been determined. The medium is rich inproteins, amino acid sugars, vitamins, minerals and growth hormones(Table 1) essential to the promotion of tissue growth. In addition,shikimic acids and quinic acids have been detected in samples of coconutwater from fruits at different stages of maturity, with the maximumamounts being found in young green coconuts. The probable role of thesealicyclic acids in romatic biosynthesis, indicates their importance inthe developing coconut. They may also play a significant role in thenutrition of plant and tissue cultures.

TABLE 1 VITAMIN, GROWTH FACTOR, SUGAR ALCOHOL AND MINERAL CONTENT OFCOCONUT WATER

COMPOUND Mg/L Nicotinic acid 0.64 Pantothenic acid 0.52 Biotin 0.02Riboflavin 0.01 Folic acid 0.003 Thiamine Trace Pyridoxime Trace Auxin0.07 Gibberellin * 1,3-Diphenylurea 5.8 Sorbitol 15.0 M-inositol 0.01Scyllo-inositol 0.05 Mg/100 g Potassium 312.0 Chloride 183.0 Sodium105.0 Phosphorous 37.0 Magnesium 30.0 Sulfur 24.0 Iron 0.10 Copper0.04 * A significant growth response was obtained from dwarf peas withan extract of 188 ml of coconut water.

The RNA-phosphorus (RNA-P) content of coconut water was found to beconsistently high at all levels of fruit maturity (Table 2). The ratioof RNA-P/DNA-P was unusually high. The role of RNA in amino acidtransport and respiratory metabolism of living cells is well known. TheRNA of coconut water would therefore effectively carry out these andother functions as part of the metabolic machinery essential to thedeveloping endosperm tissue of the coconut and therefore support thegrowth of other living cells as well, in tissue culture media.

TABLE 2 RNA-PHOSPHORUS AND DNA-PHOSPHORUS OF THE ALCOHOL-INSOLUBLERESIDUE FROM COCONUT WATER μG/mg alcohol-insoluble residue Ratio Age ofcoconuts RNA-P DNA-P RNA-P/DNA-P Young green 20.05 0.06 342.5 Mature,green 32.82 2.45 13.9

Cytokinins are a class of plant growth substances (plant hormones)active in promoting cell division. They are also involved in cell growthand differentiation and in other physiological processes. A majorcytokinin found in coconut milk was isolated using a standard procedure,the tobacco callus growth-promoting assay. The structure was determinedto be a complex trans-zeatin riboside (G3A2-ZR). The authors of thisstudy attributed at least 20% of the cytokinin activity in coconut milkto this compound².

A study that explored the efficacy of single and combined growthregulator treatments of indole-3-acetic acid (IAA), gibberelic acid(GA3) and coconut milk on plant height, yield, chlorophyll and vitamincontents of plants such as Abelmoschus esculetus L and Solanum gilo L,found that 100 mg/L GA3 and 15% coconut water were significantlyeffective treatments³.

The nutritive properties of coconut water are well recognized inOriental tradition. Immature coconuts are used as a glucose-electrolyteoral re-hydration solution for treatment of diarrhea⁴. Coconut watersolids also provide a nutritive medium for beneficial microflora in thegastrointestinal tract. A recent report also mentions the successful useof coconut water as a short-term intravenous hydration fluid⁵. Coconutwater has been used traditionally as a nutritional beverage. The FAO wasrecently granted a patent in the UK for the manufacture of bottledcoconut water that retained all nutrients. This is the first patentgiven to a UN agency. Unlike existing methods their process did not useHTST pasteurization, wherein heat treatment would inactivate some of thebiological activity of the coconut water.

Other researchers have obtained an extract from coconut water and milkcalled Cocogro, a natural product comprised of plant growth hormones andregulators, which enhances the growth of vegetables, legumes fruittrees, cereal crops, flowering plants, etc.

Green coconut milk is reported to be an effective culture medium foranimal cells (Rev Bras Biol. 1970(Apr) 30:1:97-100) and Plasmodium(Trans. R. Soc. Trop. Med Hyg. 1989 83:5, 720). A major cytokinin,G3A2-ZR, was also identified in coconut milk (Kobayashi, H: et al.Experentia, 1995 51:11 1081-84).

Although coconut milk has been used traditionally on the skin as anourishing agent, green coconut water provides unique nutrients forsustaining cell growth. For example, a protein fraction isolated fromcoconut water was absent in the milk (Birosel, DM et al. Rev. FarmBioquim Univ Sao Paulo 1976 14:35-42).

Coconut milk is used in a facemask in U.S. Pat. No. 5,660,840 and in aplant growth regulating composition in U.S. Pat. No. 5654255.

Coconut water was found to be safe for topical use. In laboratorystudies, the material is shown to have no irritating effects on theskin, and the LD₅₀ value must be greater than 2000 mg/kg whenadministered through the skin. Accordingly, coconut water is nontoxic.

Coconut water is described as an isotonic sports drink. The comparativeproperties of coconut water and conventional sports drinks are listed inTable 3⁷:

TABLE 3 SPORTS DRINKS VS. COCONUT WATER Sports drinks Coconut waterComponent (mg/100 ml) (mg/100 ml) Potassium 11.7 294 Sodium 41 25Chloride 39 118 Magnesium 7 10 Sugars 6 5

DETAILED DESCRIPTION OF THE INVENTION

An object of the invention relates to compositions containing as activeingredients, components obtained from coconut water. The compositionsare specifically formulated for use in tissue culture media, morepreferably, in supporting or culturing cells and mammalian tissuesderived from ectoderm, mesoderm and endoderm.

The composition may be used in topical applications, for example, skinand hair care formulations. The composition may also be formulated intoa nutritional supplement so as to be administered by ingestion orintravenously, thereby providing essential nutrients and repair materialfor the tissues.

The active ingredients of coconut water can be obtained byfreeze-drying, and this simple method yields a unique and stablecomposition with optimum biological activity.

The inventive composition contains optimal amounts of growth hormones(cytokinins) and RNA-phosphorus (RNA-P), an optimal ratio ofRNA-P/DNA-P, as well as desired concentrations of shikimic, quinic andindole-3-acetic acids. The inventive composition of the inventiondiffers unexpectedly from that found in raw coconut water, and a list ofits components is presented in Table 4.

TABLE 4 Composition of the invention Substance Range Total protein0.9-1.2% Carbohydrates 75-85% Sodium 350-500 mg/100 g Potassium3000-4000 mg/100 Magnesium 200-300 mg/100 g Cytokinins 0.01-0.03 mg/gIndole-3-acetic acid 0.005-0.01 mg/g Shikimic/quinic acids 25-30 Meq/mgRNA-P content 25-30 μg/g alcohol- insoluble residue RNA-P/DNA-P ratio300:1-400:1

The RNA-P/DNA-P ratio is 300:1 to 400:1. This is based on literaturereports wherein the RNA-P of 20.05 mcg/g was associated with DNA-P 0.06mcg/g of the alcohol-insoluble residue. RNA-P/DNA-P was thereforeapproximately 340:1.

Another object of the invention is a unique process for preserving thenatural growth promoting properties of green coconut water. Thenutritional properties of green coconut water are optimized by alyophilization process yielding a microbiologically stable preparationthat retains the biological activity of the ingredients. Available as apowder, the material of the invention can be conveniently stored andtransported for commercial applications. The process ensures that thebiological activity of the heat sensitive growth hormones is retained,and that the RNA phosphorus/DNA-phosphorus ratio and the amino acidcomposition are optimized for maximum biological activity.

The process employs a lyophilization technique, which preserves thebiological activity of the active ingredients. The liquid endosperm,obtained from fresh green coconuts at their peak level of maturity, isfrozen at −20° C. to −70° C. using a mixture of solid carbondioxide-acetone, followed by lyophilization in a customized lyophilizerat a reduced pressure of 0.1 to 0.8 mm Hg and a temperature of 35° C. to40° C. to produce a lyophilized amorphous material. The amorphous natureof the freeze dried material protects the protein components duringpulverization and storage. During storage, the material transforms intothe more stable crystalline state which is also less hygroscopic. Theamorphous material is pulverized under low temperature conditions toyield a powder.

The shikimic and quinic acids content and their activity in the driedpowder provide unexpected advantages over the fresh material. Theshikimic acid pathway is reported to produce salicylic acid, a knowngrowth promoter in plants. In other systems, shikimic acid and quinicacid function as potent antioxidants. Indole-3-acetic acid (plantauxin), the activity of which is retained in the dried product, has beenshown to protect mammalian cells (sperm cells).

The product can be used in topical and cosmetic formulations to promotecell growth such as in skin care and hair care formulations as well asin dry blends for nutritional and sports beverages. The invention isalso useful as an inhibitor of tumor cell proliferation in mammaliancells.

REFERENCES

-   -   1. Tulecke, W. et al. (1961) The biochemical composition of        coconut water (coconut milk)** as related to its use in plant        tissue culture. Contributions from Boyce Thompson Institute,        21:115-128.    -   2. Kobayashi, H. et al. (1995) Identification of a major        cytokinin in coconut milk. Experentia 51(11):1081-1084.    -   3. Kadiri, M. et al. (1 997) Responses of some Nigerian        vegetables of plant growth. Rev. Biol Trop. 44-45:23-28.    -   4. Adams, W. and Bratt, D E. (1992) Young coconut water for home        rehydration in children with mild gastroenteritis. Trop. Geogr.        Med. 44:149-53.    -   5. Campbell-Falck, D. et al. (2000) The intravenous use of        coconut water. Am J. Emerg. Med 18(1):108-11.    -   6. Research Report #6719 and 6720. Indian Institute of        Toxicology. May 2000. FAO: Agriculture21, New sports drink:        coconut water. October 1998.    -   7. Ma Y, Xu Q P, Sun J N, Bai L M, Guo Y J, Niu J Z Antagonistic        effects of shikimic acid against focal cerebral ischemia injury        in rats subjected to middle cerebral artery thrombosis. Chung        Kuo Yao Li Hsueh Pao 1999 Aug.;20(8):701-4    -   8. Aghil O, Bibby M C, Carrington S J, Double J, Douglas K T,        Phillips R M, Shing T K Synthesis and cytotoxicity of shikimate        analogues. Structure:activity studies based on        1-crotonyloxymethyl-3R,4R,5R-trihydroxycyclohex-2-enone.        Anticancer Drug Des 1992 Feb.;7(1):67-82    -   9. Jones R S, Ali M, Ioannides C, Styles J A, Ashby J, Sulej J,        Parke D V The mutagenic and cell transforming properties of        shikimic acid and some of its bacterial and mammalian        metabolites. Toxicol Lett 1983 Oct.-Nov.;19(1-2):43-50    -   10. Toniolli R, Bussiere J, Courot M, Magistrini M, Combarnous Y        Effect of indole-3-acetic acid (plant auxin) on the preservation        at 15 degrees C. of boar semen for artificial insemination.        Reprod Nutr Dev 1996;36(5):503-11    -   11. Nalini N, Sabitha K, Chitra S, Viswanathan P, Menon V P        Histopathological and lipid changes in experimental colon        cancer: effect of coconut kernel (Cocos nucifera Linn.) and        (Capsicum annum Linn.) red chili powder. Indian J Exp Biol 1997        September;35(9):964-71.

1. A lyophilized, nutritional composition from liquid endosperm of freshgreen coconuts (Cocos nucifera L.) comprising proteins, carbohydrates,cytokinins, ribonucleic acid-phosphorus (RNA-P), deoxyribonucleicacid-phosporous (DNA-P), indole-3-acetic acid, shikimic acid and quinicacid.
 2. The composition of claim 1, wherein the RNA-P and the DNA-P areat a ratio in a range of 300:1 to 400:1.
 3. The composition of claim 1,wherein the shikimic acid is at a concentration range of 25-30 Meq/mg.4. The composition of claim 1, wherein the quinic acid is at aconcentration range of 25-30 Meq/mg.
 5. The composition of claim 1,wherein the cytokinins are at a concentration range of 0.01-0.03 mg/g.6. The composition of claim 1, wherein the indole-3-acetic acid is at aconcentration range of 0.005-0.01 mg/g.
 7. The composition of claim 1,wherein the composition is a powder.
 8. A cosmetic formulationcomprising a composition of claim
 1. 9. A nutritional supplement for abeverage comprising a composition of claim
 1. 10. A process forobtaining a biologically active powder from fresh green coconuts (Cocosnucifera L.) comprising the steps of: a) providing liquid endosperm fromcoconuts; b) freezing the liquid endosperm using a mixture of carbondioxide and acetone to obtain a frozen liquid endosperm; c) lyophilizingthe frozen liquid endosperm of step b) to produce an amorphous material;and d) pulverizing the amorphous material of step c) to obtain thebiologically active powder.
 11. The process of claim 10, wherein theliquid endosperm is frozen at a temperature range of from at −20° C. to−70° C.
 12. The process of claim 10, wherein the lyophilizing of step c)occurs at a pressure of from 0.1 to 0.8 mm Hg and a temperature of from35° C. to 40° C.
 13. The process of claim 10, wherein the biologicallyactive powder comprises proteins, carbohydrates, cytokinins,RNA-phosphorus (RNA-P), DNA-phosporous (DNA-P), indole-3-acetic acid,shikimic acid and quinic acid.